The discovery of RNA interference (RNAi) and the major advances in the understanding of small RNA biology in the past decade have provided researchers with an invaluable tool for wide-scale and rapid genetic screening. As a research tool, RNAi takes advantage of endogenous RNA processing machinery, which permits the silencing of mRNA transcripts with complementary double stranded RNA (dsRNA). This is achieved by introduction of a so-called short interfering (si)RNA complementary to the target gene messenger RNA. Large-scale application of this technology using the robotic platforms developed for small molecule screens has led to the implementation of the first genome-wide forward genetic screens in mammalian cells. We intend to use RNAi screening technology to interrogate the mechanistic basis of immune cell responses to pathogenic insult. Our current efforts are focused on cells of the innate immune system as they form the first line of defense against numerous bacterial and viral pathogens and characterization of these initial encounters are central to our efforts to generate quantitative models of host-pathogen interactions. We are developing assays in macrophage/monocyte cell lines with both a microscopy-based 'high content'single cell readout, and also bioluminescence-based population assays using luciferase reporters driven by inflammatory gene promoters. We have designed and constructed dual promoter lentiviral vectors that permit the expression of two genes from a single virus. This has led to the creation of a RAW264.7 mouse macrophage cell line expressing two fluorescent biosensors for high content screening. The first readout expresses a GFP fusion with the RelA NFkB transcription factor driven by its endogenous promoter. This protein partitions to the cytoplasm in unstimulated cells, and translocates to the nucleus in response to a wide range of ligands that promote macrophage activation. Use of the endogenous RelA promoter facilitates a more accurate reproduction of the oscillatory cytosol/nuclear translocation previously observed with endogenous RelA. The second biosensor uses the murine TNF alpha promoter to drive expression of the red fluorescent protein mCherry, fused to a destabilizing PEST sequence which reduces the protein's half-life in the cell. This latter modification provides a more dynamic readout of TNF alpha promoter activity in kinetic experiments. We have established a clonal cell line exhibiting robust RelA translocation in the first hour, followed by a significant increase in TNF alpha promoter-driven mCherry expression after 12 hours in response to a wide range of Toll-like receptor (TLR) ligands. We have use a similar strategy to create a THP1 human monocyte cell line expressing firefly luciferase driven by the human TNF alpha promoter and also renilla luciferase driven by the ubiquitin promoter. The constitutive renilla expression provides a valuable normalization factor for cell number variability in a population-based readout. Thus, the firefly/renilla ratio in this cell line after TLR stimulation provides a measure of TNF alpha promoter activity, and we see a significant and reproducible increase in response to various TLR ligands. Interestingly, this reporter responds more quickly to LPS in this THP1 cell line (2-4 hr) than does mCherry in the RAW cell line (12-16 hr). Although the higher sensitivity of the luciferase assay may contribute to this, it is likely reflecting some difference in the response kinetics of the human and mouse TNF alpha promoters, which may have clinical relevance. Effective delivery of siRNA into hematopoietic cells remains a significant obstacle to the implementation of effective siRNA screens. It is important to establish a reproducible method that can achieve >80% knockdown (KD) of target genes in order to avoid a high frequency of false negatives in genome-wide screens. We have taken advantage of the fact that the creation of the RAW and THP1 reporter cell lines described above provide convenient control siRNA targets in the reporters they express. The ability to assay GFP and renilla luciferase KD in the RAW and THP1 lines respectively provides two advantages. First, it allows us to assay for protein rather than mRNA KD (the most common validation method for siRNA delivery), providing a more direct measure of the required endpoint needed for an effective screen. Second, the ability to run both assays in 384-well format allows for a more extensive matrix of experimental conditions which improves the chance of identifying an optimal delivery protocol. Using such an approach, this year we have completed the development of highly efficient lipid-based transfection protocols in 384-well format for both of the described RAW and THP1 cell lines. The following progress has been made this year in implementing RNAi screens in the described innate immune cell lines: - Microplate readout uniformity has been validated in both assays in 384-well format, following extensive criteria established by the NCGC small molecule screening facility. - Reproducible positive siRNA controls have been identified for a range of TLR ligands, including the TLR receptors, receptor-associated adapter proteins and protein kinases involved in the activation of the MAPK and NFkB signaling pathways. Selected controls will be included on every screening plate, to evaluate the quality metrics as the screens progress. - Pilot screens have been carried out in both cell lines to evaluate and further optimize our screening and data analysis workflows. - We have established robotic procedures for live cell screens in the RAW cell line described above. This will permit recording of both the RelA-GFP and TNF alpha promoter-mCherry readouts from the same field of cells over a 12+ hr time course. We predict that ability to score multiple phenotypes from the same cells, and to compare both single cell and population data in the same RNAi screen will be a valuable feature of our assay platform. - We have initiated genome-wide screens in both the RAW264.7 and THP1 cell lines in response to TLR ligands. In addition, we are collaborating with Scott Martin, Natasha Caplen and Chris Austin in their efforts to establish a trans-NIH RNAi screening group at the National Chemical Genomics Center. We have provided the THP1 cell line described above for the NCGC RNAi group to run parallel screens using their robotic infrastructure. This will provide an important validation of protocols in both groups, as well as increasing confidence in screen hits that overlap in each dataset.